Pronase

Pronase is recommended for primary cultures as it gives a better single cell suspension than trypsin (Gwatkin, 1973). It is not always as good with cell lines. An 0.25% solution is made by dissolving 1.25 g standard B grade pronase (Calbiochem Corp.) in 500 ml PBS-A at room temperature. After 30 min the cloudy solution is centrifuged (100 g, 10 min) and sterilised by filtration as for trypsin. 

Collagenase is reported to cause least damage and is used in the preparation of clonal cell cultures (Hilfer, 1973). 

Combined use of collagenase and hyaluronidase followed by trypsin or pronase without vigorous agitation (which leads to loss of cell viability) was satisfactory for mouse mammary gland cells (Prop and Wiepjes, 1973). 

Preparations of collagenase vary. Worthington (Appendix 3) supply four varieties of crude collagenase, each of which is suited to isolation of cells from a particular tissue. Individual batches of enzyme can be reserved and tested. 

Dispase is a neutral protease from Bacillus polymyxa available from Boehringer Corp. Ltd. It is particularly suitable for disaggregation of animal tissues, which seldom suffer from prolonged treatment. It is available alone, or mixed with collagenase. It requires Ca2+ for activity and its action is readily neutralised by EDTA. 

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The neuraminidase molecule is a homotetramer made up of four identical polypeptide chains, each of around 470 amino acids the exact number varies depending on the strain of the virus. If influenza virus is treated with the proteolytic enzyme pronase, the head of the neuraminidase, which is soluble, is cleaved off from the stalk projecting from the viral envelope. The soluble head, comprising four subunits of about 400 amino acids each, can be crystallized. 

Error 1 CRMs completely or partly not identical with the matrix to be analyzed Ber-mejo-Barrera et al. (1999) studied enzymatic hydrolysis procedures using pronase E as sample pretreatment for multi-element - Cu, Fe, Mg, Zn, Ag, As, Cd, and Pb -determination in biological materials, mussel samples and human hair. 

Bermejo-Barrera P, I Ernanuez-Nocelo S, Moreda-PiSeiro A and Bekmejo-Barrera A (1999) Useftilness of enzymatic hydrolysis procedures based on the use of pronase E as sample pre-treatment for multi-element determination in biological materials. J Anal At Spcctrom 14 1893-1900. 

The effects of various enzymes on the activity of HPLC fractions that inhibited 3H-PCP binding were investigated. As shown in table 1, pronase (0.5 pg/ml), carboxypeptidase A (0.1 unit/ml), and trypsin (3.0 g/ml ) markedly decreased the potency of 10 n units of PCP-like activity. No significant change in activity was. seen when fractions were incubated with alpha-chymotrypsin. 

Nasr SH, Galgano SJ, Markowitz GS, et al. Immunofluorescence on pronase-digested paraffin sections a valuable salvage technique for renal biopsies. Kidney Int. 2006 70 2148-2151. 

For the proteolytic digestion procedure, dissolve pronase in water at a concentration of 1 percent (w/v). 

If pronase digestion of the biotinylated protein is to be done, heat 100 pi of the sample at 56°C for 10 minutes, then add 10 pi of the pronase solution. Allow the sample to digest enzymatically at room temperature overnight. If no pronase digestion is desired, simply use the biotinylated protein solution prepared in step 3 without further treatment. 

Recently, sulfur mustard has been shown to alkylate a cysteine residue in human serum albumin (10). The site of alkylation was identified in a tryptic digest of albumin from blood exposed to [14C]sulfur mustard. A sensitive method for its analysis was developed based on Pronase digestion of alkylated albumin to the tripeptide S-[2-[(hydroxyethyl)thio]ethyl-Cys-Pro-Phe, and detection using micro-LC-MS-MS. In vitro exposure of human blood to > 10 nM sulfur mustard could be detected employing this method. The analytical procedure was successfully applied to albumin samples from Iranian casualties of the Iraq-Iran war. 

The reactivity of compounds such as 28 was clearly demonstrated by the peroxidase-catalyzed covalent binding of A -methyW-hydroxyellipticine (27) to proteins (756). Using horseradish peroxidase and hydrogen peroxide, tritiated-27 was converted to the 9-oxoellipticine derivative in the presence of bovine serum albumin (BSA) and human antibovine IgG in vitro. Covalent binding to these proteins was confirmed by gel electrophoresis, combustion, and liquid scintillation analysis. Dissolution of the BSA-ellipticinium derivative with pronase and... 

Dissolve 0.1 g pronase in calcium chloride solution, and adjust pH to 7.8 with 0.1 M sodium hydroxide. 

Place sections in pronase solution at room temperature, and incubate for predetermined optimum time (approximately 10 min). 

Figure 3.10. Effect of pronase on FcyRII (CD32) and Fc yRIII (CD16) expression on neutrophils. Neutrophils were incubated in the absence (-) or presence (+) of pronase (50 pg/ml) for 30 min at 37 °C. After this incubation, expression of FcyRII and FcyRIII was determined by FACS analysis.

In resting neutrophils, about 50% of the total cellular FcyRIII pool is expressed on the cell surface. There is considerable variation in this value because many methods used to isolate neutrophils can also inadvertently mobilise these subcellular receptors. The remainder of the total cellular FcyRIII that is not expressed on the plasma membrane is present in the subcellular pool. However, if the FcyRIII normally present on the plasma membrane is cleaved (e.g. via the action of elastase or pronase) and the cells subsequently activated, then FcyRIII reappears on the cell surface via the mobilisation of these pools. Thus, the expression can be restored to up to 70% of the resting level within 15 min via such a translocation. During activation (and presumably priming), FcyRIII (together with other plasma membrane markers) is also translocated to the plasma membrane however, because the receptor is also shed from the cell, the total number of receptors on the cell surface remains largely unchanged. There is also some evidence that continued expression of FcyRIII on the cell surface requires de novo biosynthesis of this receptor (see Fig. 7.8). 

See Section IV.1 for alternative methods of chiral resolution. Partial chemical hydrolysis of proteins and peptides with hot 6 M HC1, followed by enzymatic hydrolysis with pronase, leucine aminopeptidase and peptidyl D-amino acid hydrolase, avoids racemiza-tion of the amino acids281. The problems arising from optical rotation measurements of chiral purity were reviewed. Important considerations are the nonideal dependence of optical rotation on concentration and the effect of chiral impurities282. 

Figure 3.18. Effect of pronase on the CHEQ of probe 13 with 50 equivalents of poly-L-glutamate. a, 10 units pronase , 5 units pronase , 1 unit pronase o, no pronase.

As part of an extensive investigation, a large variety of /3-peptides, plus some y-peptides, were tested against proteinase K and pronase [228], The substrates included shorter and longer /33-peptides, /32-pcplides, and a few y-... 

Protease treatment After washing, cells are exposed to a solution of 0.04% protease (pronase E) in Ca TMg free HEPES-containing ethylene glycol tetra acetic acid (EGTA) (Sigma) for 15 minutes at 37°C as applied in (93). Cells are then washed three times with ice-cold phosphate buffered saline (PBS) containing 0.1% BSA. 

Though the contractions caused by cotton dust and cotton bract have important differences, there are also similarities between their chemical properties. It is doubtful that either of the active agents are proteins because pronase has no affect on the extracts and they are labile even at 4 C. In addition, both active substances were extracted with butanol (69). 

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